viruses Communication Genome Sequence Analysis of First Ross River Virus Isolate from Papua New Guinea Indicates Long-Term, Local Evolution Alice Michie 1, John S. Mackenzie 2,3,4 , David W. Smith 2 and Allison Imrie 1,* 1 School of Biomedical Sciences, University of Western Australia, Nedlands, WA 6009, Australia; [email protected] 2 PathWest Laboratory Medicine Western Australia, Nedlands, WA 6009, Australia; [email protected] (J.S.M.); [email protected] (D.W.S.) 3 School of Chemistry and Molecular Biosciences, University of Queensland, St. Lucia, QLD 4072, Australia 4 Faculty of Health Sciences, Curtin University, Bentley, WA 6102, Australia * Correspondence: [email protected] Abstract: Ross River virus (RRV) is the most medically significant mosquito-borne virus of Australia, in terms of human morbidity. RRV cases, characterised by febrile illness and potentially persistent arthralgia, have been reported from all Australian states and territories. RRV was the cause of a large- scale epidemic of multiple Pacific Island countries and territories (PICTs) from 1979 to 1980, involving at least 50,000 cases. Historical evidence of RRV seropositivity beyond Australia, in populations of Papua New Guinea (PNG), Indonesia and the Solomon Islands, has been documented. We describe the genomic characterisation and timescale analysis of the first isolate of RRV to be sampled from PNG to date. Our analysis indicates that RRV has evolved locally within PNG, independent of Australian lineages, over an approximate 40 year period. The mean time to most recent common Citation: Michie, A.; Mackenzie, J.S.; ancestor (tMRCA) of the unique PNG clade coincides with the initiation of the PICTs epidemic in Smith, D.W.; Imrie, A. Genome mid-1979. This may indicate that an ancestral variant of the PNG clade was seeded into the region Sequence Analysis of First Ross River Virus Isolate from Papua New Guinea during the epidemic, a period of high RRV transmission. Further epidemiological and molecular- Indicates Long-Term, Local Evolution. based surveillance is required in PNG to better understand the molecular epidemiology of RRV in Viruses 2021, 13, 482. https:// the general Australasian region. doi.org/10.3390/v13030482 Keywords: arbovirus; mosquito-borne disease; alphavirus; Papua New Guinea; Australia Academic Editors: Bas B. Oude Munnink, Marietjie Venter and Luisa Barzon 1. Introduction Received: 15 February 2021 In Australia, the most common mosquito-borne viral infection is caused by Ross Accepted: 12 March 2021 River virus (RRV), an alphavirus of the family Togaviridae [1]. Disease associated with RRV Published: 15 March 2021 infection is characterised by potentially persistent and debilitating fatigue, myalgia, and arthralgia and/or arthritis. RRV cases have been reported from all Australian states and Publisher’s Note: MDPI stays neutral territories since it was first isolated from Aedes vigilax mosquitoes sampled in far-north with regard to jurisdictional claims in Queensland in 1959 [2]. published maps and institutional affil- iations. RRV is currently considered to be both a mosquito vector species and amplifying host generalist, with macropods (such as wallabies and kangaroos) considered the major reservoir host for RRV maintenance and transmission [3]. Humans have been implicated as short-term amplifying hosts during disease epidemics including the large-scale, virgin-soil epidemic that occurred across multiple Pacific Island countries and territories (PICTs) from Copyright: © 2021 by the authors. 1979 to 1980 [4–6]. Cases were reported from Fiji, American Samoa, the Cook Islands, Licensee MDPI, Basel, Switzerland. New Caledonia and Wallis and Futuna, with disease suspected in northern Tonga [7]. This article is an open access article Samoa did not report cases during this time, but during an April 1980 investigation of a distributed under the terms and conditions of the Creative Commons dengue outbreak, high titres of anti-RRV haemagglutination inhibition (HI) antibody were Attribution (CC BY) license (https:// detected. RRV activity likely occurred in Samoa during the 1979–1980 outbreak but went creativecommons.org/licenses/by/ undetected [7]. Viraemic travelers are the suspected mode of rapid spread between these 4.0/). adjacent island nations [8]. Viruses 2021, 13, 482. https://doi.org/10.3390/v13030482 https://www.mdpi.com/journal/viruses Viruses 2021, 13, 482 2 of 8 RRV transmission seemingly ceased in the region in mid-1980, presumably due to the lack of efficient reservoir hosts in the area [7,8]. A viraemic traveler from eastern Australia has been postulated as the source of the outbreak, which began in Fiji in April 1979, based on sequence similarity of epidemic viruses to the ‘Eastern-Australian’ lineage of RRV [9]. The geographical basis of RRV lineage nomenclature was not supported in our recent, more comprehensive genome-scale RRV phylogenetic analysis, where we showed that virus isolates that clustered as distinct lineages were geographically dispersed and sampled Australia wide [10]. The exact origins of the PICTs epidemic is now less certain, given viruses similar to those sampled during the epidemic, were sampled across Australia between 1982 and 1988 [10]. Four distinct genotypes of RRV (G1–4) have been characterised, with G4 in contemporary circulation across Australia [10]. RRV seropositivity has been described in the adult and general population of many regions of Papua New Guinea (PNG), sampled from 1960 to 1969, as well as regions of In- donesia and the Solomon Islands [11]. A later study found a 59% RRV seroprevalence in the Southern Highlands of PNG in 1991, and endemicity was suggested by increasing antibody prevalence with age [12]. RRV as a cause of arthritis in PNG has been confirmed [13,14]. RRV has been isolated from several mosquito species within Australia that are also present in PNG including Ae. vigilax, Ae. notoscriptus and Mansonia uniformis [15,16]. The exact prevalence, distribution, and genetic diversity of RRV in PNG is currently unknown, due to the lack of comprehensive surveillance in the region [17]. RRV cases were not reported from PNG during the PICTs epidemic. The first and only isolation of RRV from PNG to date was made in 1997 from a pool of Anopheles farauti mosquitoes collected at Wandoo village in the Bensbach region of the Western Province [16]. In this study, the genome sequence of this PNG isolate was derived and analysed with available RRV genome sequences to better understand the molecular epidemiology of RRV in the greater Australasian region. 2. Materials and Methods 2.1. Virus Isolation, RNA Extraction and Next Generation Sequencing The virus isolate, PNG3075, was isolated from a pool of An. farauti mosquitoes trapped in 1997 from the Bensbach locality of the Western Province of PNG [16]. The isolate was identified as RRV using a tissue culture-based fixed cell enzyme immunoassay involving specific, identifying monoclonal antibodies on fixed C6/36 monolayers (Ae. albopictus larvae, ATCC® CRL-1660™)[16,18]. The isolate was not investigated on a molecular level at the time of its initial characterisation. PNG3075 was amplified in the present study through a single passage on Vero cells (African Green Monkey kidney epithelium, ATCC® CCL-81™) maintained in Dulbecco’s Modified Eagle Media (Gibco DMEM, Thermo Fisher Scientific, Waltham, Massachusetts, USA) supplemented (by volume) with 5% heat-inactivated fetal bovine serum (FBS), 1% L- glutamine (Life Technologies, Carlsbad, California, USA) and 1% penicillin/streptomycin (Life Technologies). The Roche High Pure RNA extraction kit (Roche, Basal, Switzer- land) was utilised as per the manufacturers protocol from viral supernatant, concentrated through ultra-centrifugation in Millipore Amicon Ultra-15 centrifugal units (Merck Milli- pore, Darmstadt, Germany). The TruSeq stranded mRNA kit (Illumina, San Diego, California, USA) was used for library preparation. Superscript III reverse transcriptase (Invitrogen, Waltham, Mas- sachusetts, USA) was used for cDNA synthesis. Libraries were validated using the Agilent 1000 DNA kit (Agilent Technologies, Santa Clara, California, USA) and subsequently nor- malized and pooled. Sequence data were generated on the MiSeq platform with the MiSeq Micro v2 kit (Illumina). Paired-end reads of 150 bp were demultiplexed and assessed for quality within the FastQC v0.11 program [19], and subject to de novo genome sequence generation within CLC Genomics Workbench v7.5 (QIAGEN, Hilden, Germany) to derive a full-coding contiguous sequence, 11,837 nucleotides in length. A total of 567,724 reads Viruses 2021, 13, 482 3 of 8 were generated for the PNG3075 sample, of which 86.89% (493,294 reads) were used to generate the complete coding PNG3075 sequence. 2.2. Phylogenetic Analysis The derived genome was aligned with 104 publicly available RRV genome sequences using MAFFT 7.338 within Geneious 11.1.4 [20]. Most available sequence data were derived in our recent RRV phylogenetic investigation, with isolates primarily sampled in Western Australia [10]. For phylogenetic analysis, sequences were manually trimmed to remove 50 and 30 untranslated regions (UTRs) and then realigned. A maximum likelihood phylogeny was reconstructed using RAxML 8.2.11 with 1000 bootstrap replicates within Geneious 11.1.4, under a GTR+G+I nucleotide substitution model—the best-suited model according to JModelTest 2 analysis [21,22]. All phylogenies were visualized and edited within FigTree v1.4.4 [23]. 2.3. Temporal